The vertebrate isoactins are a multigene family of closely related proteins which are differentially expressed during development. Two cytoplasmic actins are found in nonmuscle cells and unique actins are associated with the four major muscle types: skeletal, cardiac, vascular and enteric. Most of these tissues express more than one muscle actin at some developmental stage. However, neither the cellular nor the molecular basis for the developmental expression of multiple actins is known because of the difficulty in discriminating among these isoforms by biochemical or immunochemical criteria. This proposal has two specific aims which employ cytological methodologies to address these issues by localizing individual isoactins and their mRNAs in the developing rat and in cultured muscle cells. First, we propose to monitor the expression and distribution of each isoactin using immunocytochemical methods. This approach will make use of several muscle selective polyclonal and monoclonal antibodies to actin that have been developed in this laboratory. We also will prepare additional antibodies that react selectively with peptides containing unique isoactin sequences. The ontogeny and cellular distribution of the isoactins will be followed in the developing rat embryo and neonate. Tissue culture cells will be used for clonal analyses and to examine the intracellular distribution of the isoactins. Second, the tissue and cellular expression of each isoactin mRNA will be determined using in situ hybridization with labeled oligonucleotide sequences that are complementary to unique portions of each isoactin mRNA. Specific cDNA probes containing unique nucleotide sequences within the 3'-untranslated regions of the mRNAs for cardiac, skeletal and cytoplasmic actins are available. These experiments should provide new and important information concerning the cellular basis for the developmental expression of multiple actins in the muscle. It will be possible to determine whether all cells express each of the isoactins or heterogeneity exists in the expression of the actins among cells within a tissue. Moreover, if multiple isoactins are expressed in the same cell, it will be possible to determine whether they are similarly distributed in cells during myogenesis or unique isoactin distributions are present implying architectural regulation and functional specialization.